Like other members of the picornavirus family, hepatitis A virus (HAV, genus hepatovirus) contains a single-stranded, positive-sense RNA genome with a lengthy (735 nucleotide [nt]) 5' nontranslated region (5'NTR). The 5'NTR of HAV (HM175 strain) contains an internal ribosomal entry site (IRES) located between nt 152 and nt 735, which regulates cap-independent, internal initiation of translation of the viral polyprotein, although the level of efficiency with which the HAV IRES promotes translation is much less than that of other picornavirus IRES elements both in vivo and in vitro (E. Brown, A. Zajac, and S. Lemon, J. Virol. 68:1066-1074 (1994), L. Whetter, et al., J. Virol. In press (1994)). The 5'NTR of HAV may possibly have other functions which are essential for virus replication, including control of positive-strand RNA synthesis and possibly encapsidation (R. Andino et al., Cell 63:369-380 (1990)).
Although there are substantial differences between the predicted secondary structures of the 5'NTRs of HAV and all other picornaviruses, the secondary structure of the HAV 5'NTR more closely resembles that of the cardioviruses and aphthoviruses than the corresponding structure in rhinoviruses and most enteroviruses (E. Brown, et al., J. Virol. 65:5828-5838 (1991)). Among other similarities, the 5'NTRs of hepatoviruses, cardioviruses, and aphthoviruses share the potential to form two or more pseudoknots in the noncoding region upstream of the IRES element (E. Brown, et al., J. Virol. 65:5828-5838 (1991), E. Brown, A. Zajac, and S. Lemon, J. Virol. 68:1066-1074 (1994), B. Clarke, et al., Nucleic Acids Res. 15:7066-7079 (1987), S. Jang, et al., J. Virol. 63:1651-1660 (1989), R. Kuhn, N. Luz, and E. Beck, J. Virol. 64:4625-4631 (1990), C. Pleij, Proc. VIII Int. Congr. Virol. 49-50 (1990)). Also present in this region is a pyrimidine-rich sequence which consists of an almost pure polycytidylic acid tract in the cardioviruses and aphthoviruses. In the hepatoviruses, the corresponding region contains a mixture of uridylic acids and cytidylic acids (in a 24:14 ratio in the HM175 strain of HAV), with only two purines located within a 40-nt-long, nearly pure polypyrimidine tract (pY1 domain, nt 99 to 138). In each of these virus genera, this pyrimidine-rich tract appears to separate two discrete regions of RNA secondary structure (E. Brown, et al., J. Virol. 65:5828-5838 (1991), B. Clarke, et al., Nucleic Acids Res. 15:7066-7079 (1987)). A similarly located pyrimidine-rich tract is not found in either the enterovirus or rhinovirus 5'NTR.
There are other pyrimidine-rich tracts within the 5'NTR of HAV (K. Chang, E. Brown, and S. Lemon, J. Virol. 67:6716-6725 (1993)), but the pY1 domain is the lengthiest and most prominent of these regions. Although considerable sequence heterogeneity exists within this domain among different human hepatoviruses (E. Brown, et al., J. Virol. 65:5828-5838 (1991)), the general features of this domain are conserved among all strains of HAV. A striking aspect of the pY1 domain, which is unique to the HAV 5'NTR among all other picornaviral 5'NTRs, is the presence of tandem repeats of the sequence motif (U)UUCC(C). Curiously, this motif closely resembles the core sequence of the "box A" motif of Pilipenko et al. (E. Pilipenko, et al., Cell 68:119-131 (1992)), which is present in a conserved location in all picornaviruses, about 20-25 nt upstream of the initiator AUG and which may play an important role in internal initiation of translation.
Previous modeling of the secondary structure of the 5'NTR of HM175 virus predicted that the pY1 domain and the immediately adjacent sequence from nt 139 to nt 154 were likely to be single-stranded (E. Brown, et al., J. Virol. 65:5828-5838 (1991)).